Sample: CBA.G4F-426-L3W

Analysis Parameters

Analytes Alpha1 AntiTrypsin, Haptoglobin, Transferrin
Equipment Protocol Vertical Tanks 1
Analysis Protocol V1.01
Usage Research Use Only

Summary

The analysis presented in this document resulted from following the biosignatures ‘Gel-As-Assay’ workflow. A hybrid 2D DiGE approach is used to increase automation, quality and reproducibility. The key difference is that a fixed standard is always used rather than a pooled standard.

2µL Human Plasma is labelled with Cy5 (or equivalent) and a fixed standard labelled with Cy3 following protocols defined by biosignatures. Images of the channels are then uploaded to a cloud analysis platform. The Cy3 standard is then aligned into a fixed reference space and automated QC procedures assess the fixed standard. If the standard passes the automated procedures a fixed feature pattern is applied and feature measures obtained. This report is then automatically generated detailing the results for a selected subset of analytes.

summary table of results

Analytes:

Overview

The gel image below shows the locations of the analyte chains presented in this report.

Alpha-1-antitrypsin (P01009)

From: Human plasma protein N-glycosylation

Alpha-1-antitrypsin (AAT), also known as alpha-1-protease inhibitor, alpha-1-antiproteinase or serpin A1, consists of 418 amino acids (including a 24 amino acid signal peptide) with an apparent mass of 51 kDa (including glycosylation). It is mainly produced in the liver by hepatocytes, but is also synthesized in monocytes, intestinal epithelial cells, and in the cornea [52, 208–211]. Due to its small size and polar properties, the glycoprotein can easily move into tissue fluids [52]. In healthy individuals, a plasma level of approximately 1.1 mg/mL is found, but the concentration can increase three- to four-fold during inflammation [212–215]. AAT occurs as three different amino acid sequences, of which the first is set as the standard sequence. Form 2 differs in the amino acid sequence 356–418 and form 3 lacks the amino acid sequence 307–418.

CBA.G4F-426-L3W A1AT_1

Replicate 1 : 50014_Cy5 450_Cy3 455 Cy5.gel_sc.gel

Replicate 2 : 72288_Cy5 440_Cy3 455 Cy5.gel_sc.gel

Replicate 3 : 78543_Cy5 440_Cy3 445 Cy5.gel_sc.gel

SF0202 SF0210
50014_Cy5 450_Cy3 455 Cy5 8.666647 8.598220
72288_Cy5 440_Cy3 455 Cy5 8.709795 8.649273
78543_Cy5 440_Cy3 445 Cy5 8.703673 8.648222

CBA.G4F-426-L3W A1AT_2

Replicate 1 : 50014_Cy5 450_Cy3 455 Cy5.gel_sc.gel

Replicate 2 : 72288_Cy5 440_Cy3 455 Cy5.gel_sc.gel

Replicate 3 : 78543_Cy5 440_Cy3 445 Cy5.gel_sc.gel

SF0514 SF0520 SF0527 SF0530 SF0532
50014_Cy5 450_Cy3 455 Cy5 9.647821 10.15144 10.11026 9.504948 9.584590
72288_Cy5 440_Cy3 455 Cy5 9.736252 10.22630 10.18388 9.604677 9.673697
78543_Cy5 440_Cy3 445 Cy5 9.605014 10.07807 10.05247 9.473243 9.509407

CBA.G4F-426-L3W A1AT_3

Replicate 1 : 50014_Cy5 450_Cy3 455 Cy5.gel_sc.gel

Replicate 2 : 72288_Cy5 440_Cy3 455 Cy5.gel_sc.gel

Replicate 3 : 78543_Cy5 440_Cy3 445 Cy5.gel_sc.gel

SF0557 SF0558 SF0575 SF0584 SF0581
50014_Cy5 450_Cy3 455 Cy5 8.259199 8.285513 8.226841 8.214465 8.237215
72288_Cy5 440_Cy3 455 Cy5 8.268219 8.296546 8.222285 8.209036 8.234034
78543_Cy5 440_Cy3 445 Cy5 8.355850 8.377701 8.315077 8.296048 8.316545

Haptoglobin (P00738)

From: Human plasma protein N-glycosylation

Haptoglobin (Hp) is a 406 amino acid (18 amino acid signal peptide) acute-phase glycoprotein with a peptide backbone of 45 kDa. It is synthesized in the liver by hepatocytes as a single polypeptide chain and is also found in skin [304, 305]. During its synthesis, Hp is cleaved into a light a chain and a heavy ß chain that are connected via disulfide bonds. Two variants of the a chain originating from the sequence Val19-Gln160 and differing by the subsequence Glu38-Pro96 can exist, a1 having this subsequence once while a2 has it twice, resulting in a chains of 83 or 142 amino acids with a respective molecular mass of 9 and 16 kDa. The 40 kDa ß chain is made of 245 amino acids originating from the sequence Ile162-Asn406 [306, 307]. The combination of different allelic variants of the a chain (a1 and a2) with ß chain(s) creates the polymorphism observed in Hp. There are three major Hp phenotypes called Hp1-1, Hp2-1 and Hp2-2. They have a configuration of (a1ß)2, (a1ß)2 + (a2ß)n = 0, 1, 2, … and (a2ß)n = 3, 4, 5, …, respectively, which are observed at different ratios among ethnicities [118, 308–310]. Caucasians have around 13 % of phenotype Hp1-1, 46 % of Hp2-1 and 41 % of Hp2-2. Hp is typically found at a plasma levels in the range of 0.6–2.3 mg/mL with a mean of 1.32 mg/mL [118]. Elevated Hp levels have been reported with inflammation and malignant diseases [308, 311, 312]. It should be taken into account that the concentration as well as the molecular mass including glycosylation may vary among phenotypes (86–900 kDa) [118]. The half-life of Hp is found to be on average four days.

The major function of Hp is to protect tissues from oxidative damage by capturing hemoglobin [307, 313]. It has been reported that Hp polymorphism has an effect on its physiological properties, for instance Hp1-1 binds hemoglobin stronger than Hp2-2 [314]. Certain diseases seem to be dependent on the polymorphism, as individuals with the Hp1-1 phenotype seem to have a higher concentration of induced antibodies in their plasma after vaccination, infections or liver diseases compared to the other phenotypes [118, 310].

CBA.G4F-426-L3W HPT.alpha.

Replicate 1 : 50014_Cy5 450_Cy3 455 Cy5.gel_sc.gel

Replicate 2 : 72288_Cy5 440_Cy3 455 Cy5.gel_sc.gel

Replicate 3 : 78543_Cy5 440_Cy3 445 Cy5.gel_sc.gel

SF0832 SF0836 SF0842
50014_Cy5 450_Cy3 455 Cy5 9.540723 9.596079 8.598589
72288_Cy5 440_Cy3 455 Cy5 9.695109 9.513404 8.618305
78543_Cy5 440_Cy3 445 Cy5 9.544381 9.425613 8.649974

CBA.G4F-426-L3W HPT.Beta.

Replicate 1 : 50014_Cy5 450_Cy3 455 Cy5.gel_sc.gel

Replicate 2 : 72288_Cy5 440_Cy3 455 Cy5.gel_sc.gel

Replicate 3 : 78543_Cy5 440_Cy3 445 Cy5.gel_sc.gel

SF0620 SF0634 SF0627 SF0642 SF0654 SF0665 SF0648 SF0668 SF0666 SF0671 SF0685 SF0690 SF0689 SF0683
50014_Cy5 450_Cy3 455 Cy5 8.380686 9.279120 8.912608 9.483188 9.581490 9.693692 8.719971 8.661467 9.305014 8.239065 8.295549 8.728426 8.692826 8.583168
72288_Cy5 440_Cy3 455 Cy5 8.472405 9.374668 9.265586 9.555985 9.657395 9.796292 8.704668 8.733272 9.442959 8.251664 8.292549 8.750683 8.740817 8.614864
78543_Cy5 440_Cy3 445 Cy5 8.420682 9.203819 8.822912 9.428512 9.541297 9.661989 8.725183 8.731821 9.335121 8.333511 8.402456 8.759041 8.732466 8.626765

Transferrin (P02787)

From: Human plasma protein N-glycosylation

Serotransferrin (STF), also known as transferrin, ß1 metal binding globulin or siderophilin, is a 698 amino acid protein (19 amino acids of which are signal peptide) with a molecular mass of approximately 77 kDa (without glycosylation) [8, 337]. The protein consists of two globular domains, the N-lobe and the C-lobe which divided into two subdomains each (N1, N2, C1 and C2). The two main domains are connected by a short linker peptide [337–339]. The N-lobe is 336 amino acids in size and spans from Val25 to Glu347, while the C-lobe is 343 amino acids long and ranges from Val361 to Lys683 [337]. The lobes can interact to form a hydrophilic metal ion binding site [337]. STF is mostly produced by hepatocytes, although other tissues have also shown expression, albeit at significantly lower amounts [337]. The plasma concentration is highly stable from the age of 2 years on, with a range between 2 and 3 mg/mL [337, 340]. Levels may increase during pregnancy up to 5 mg/mL [141].

CBA.G4F-426-L3W TRFE

Replicate 1 : 50014_Cy5 450_Cy3 455 Cy5.gel_sc.gel

Replicate 2 : 72288_Cy5 440_Cy3 455 Cy5.gel_sc.gel

Replicate 3 : 78543_Cy5 440_Cy3 445 Cy5.gel_sc.gel

SF0357 SF0359 SF0372 SF0373 SF0383 SF0387 SF0384 SF0381
50014_Cy5 450_Cy3 455 Cy5 8.758255 9.254740 8.980550 9.466222 9.955700 10.21983 9.583420 8.479699
72288_Cy5 440_Cy3 455 Cy5 8.951699 9.392995 9.112507 9.631548 10.051131 10.28042 9.731512 8.589328
78543_Cy5 440_Cy3 445 Cy5 8.901775 9.190750 9.012377 9.322508 9.790879 10.00170 9.334150 8.472823

Statistical Process Control

From: An introduction to statistical process control in research proteomics

Background: Statistical process control is a well-established and respected method which provides a general purpose, and consistent framework for monitoring and improving the quality of a process. It is routinely used in many industries where the quality of final products is critical and is often required in clinical diagnostic laboratories [1,2]. To date, the methodology has been little utilised in research proteomics. It has been shown to be capable of delivering quantitative QC procedures for qualitative clinical assays [3] making it an ideal methodology to apply to this area of biological research.

Control charts and possibly image areas from the standard.

Audit Logs:

 Run overview

Run # 208
Sample / Study Human plasma (Lot: SLCC1673)/ NUBZ64100, NUCX182416, NUBZ648698, NUAQ387360, NUFH402412, NUFT865086, NUFR670624, NUFT766262, NUFU715690, NUFT770122, NUFT766280, NUCX210223
Completion (Scan) Date 25/09/2020
Comments OOD GE strips, biotium dye, sample load 2uL. Pre-mix buffers. Standard IEF with cuploading. Standard 2D with type 2 water. 4% equilibration buffer.

 Labelling

Technician:EA & NH

Sample: 2µL Human Plasma

Solution / reagent Batch / Lot # Reference
DIGE labelling buffer 6 LB-RS-06
Tris-HCl pH 8.5 2 LB-RS-02
DMF STBH7014
Cy3 (Biotium) 13C1212
Cy5 (Biotium) 18C0626-1075
Lysine 4 LB-RS-07
2x DIGE buffer 6 LB-RS-04

 Iso Electric Focusing

Technician:EA & NH

Solution / reagent Batch / Lot #
1x DIGE buffer 14
Control sample Human plasma/ SLCC1673
IPG strips 10277023
Reswell Date 22/09/2020
Time strip 1 16:10:00
Time oil added 16:40:00
Equipment ID #
Reswell tray 3
IPGphor unit 1

Transfer to IPGphor: 23/09/2020 10:54:00

Technician: EA & NH

# Control ID (Cy3) Sample ID (Cy5) Strip ID IEF Tray # Run Time
1 Human plasma/ SLCC1673 NUBZ640100 49844 2 23.5
2 Human plasma/ SLCC1674 NUCX182416 49845 2 23.5
3 Human plasma/ SLCC1675 NUBZ648698 49846 2 23.5
4 Human plasma/ SLCC1676 NUAQ387360 49847 2 23.5
5 Human plasma/ SLCC1677 NUFH402412 49848 2 23.5
6 Human plasma/ SLCC1678 NUFT865086 49849 2 23.5
7 Human plasma/ SLCC1679 NUFR670624 49850 2 23.5
8 Human plasma/ SLCC1680 NUFT766262 49851 2 23.5
9 Human plasma/ SLCC1681 NUFU715690 49852 2 23.5
10 Human plasma/ SLCC1682 NUFT770122 49853 2 23.5
11 Human plasma/ SLCC1683 NUFT766280 49854 2 23.5
12 Human plasma/ SLCC1684 NUCX210223 49855 2 23.5

 2D SDS


Casting (A2DE optimiser)

Technician: NH

Solution / reagent Batch / Lot # Weight (g) Reference
Tris-SDS 24 LB-RS-11
Glycerol 17 LB-RS-09
Acrylamide 07-19-22 WI-LB-001 5.2.1(1)
APS MKCG5404 WI-LB-001 5.2.1(3a)
TEMED STBH7073 WI-LB-001 5.2.1(3b)
Equipment Set
Gel plates: 1
Casting box: 1
Optimiser: 1

Second dimension run properties (2D Tank1)

Technician: EA & NH

Event Time W mA V
Start 15:32:00 8 170 46
Duration 17h30
Power up 09:02:00
Time off 09:18:00 25 190 130
Total time 17h 46m

 Imaging

Technician: EA & NH

Imager: Typhoon 9400

Calibration App Log File

The imager was calibrated on 25/09/2020 following protocol P-212 and Calibration App Version 1.01.
Dye channel PMT
Cy5 450
Cy3 455

Scanner Driver App

The gels were imaged in pairs using Scanner Driver App version 1.01. The app automatically cropped and rotated the scans. Scans were performed at 100 and 200 micron resolutions. Cy3 and Cy5 channels were imaged simultaneously.

Whole gel visual review

The gels passed a visual review (under OP-28) before upload to the cloud for processing.

Scanning App Log File

Opening excel file: A:\Lab\GelRunning\Runs\0208\Run 208.xlsx

Loaded scans:

Strip1: 49844 Strip2: 49845

Strip1: 49846 Strip2: 49847

Strip1: 49848 Strip2: 49849

Strip1: 49850 Strip2: 49851

Strip1: 49852 Strip2: 49853

Strip1: 49854 Strip2: 49855

Starting scan of Strip1: 49844 Strip2: 49845

Finished scan of Strip1: 49844 Strip2: 49845

Scanned images in 00:31:26.9429270:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49844_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49844_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49845_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49845_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49844_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49844_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49845_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49845_Cy5 560_Cy3 590 Cy5.gel

File 49844_Cy5 560_Cy3 590 Cy3.gel had 77362 saturated pixels of 5304000

File 49845_Cy5 560_Cy3 590 Cy3.gel had 107383 saturated pixels of 5304000

Starting scan of Strip1: 49846 Strip2: 49847

Finished scan of Strip1: 49846 Strip2: 49847

Scanned images in 00:31:16.6743397:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49846_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49846_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49847_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49847_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49846_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49846_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49847_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49847_Cy5 560_Cy3 590 Cy5.gel

File 49846_Cy5 560_Cy3 590 Cy3.gel had 99403 saturated pixels of 5304000

File 49847_Cy5 560_Cy3 590 Cy3.gel had 85804 saturated pixels of 5304000

Starting scan of Strip1: 49848 Strip2: 49849

Finished scan of Strip1: 49848 Strip2: 49849

Scanned images in 00:31:19.2494869:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49848_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49848_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49849_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49849_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49848_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49848_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49849_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49849_Cy5 560_Cy3 590 Cy5.gel

File 49848_Cy5 560_Cy3 590 Cy3.gel had 97777 saturated pixels of 5304000

File 49849_Cy5 560_Cy3 590 Cy3.gel had 61913 saturated pixels of 5304000

Starting scan of Strip1: 49850 Strip2: 49851

Finished scan of Strip1: 49850 Strip2: 49851

Scanned images in 00:31:20.2025415:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49850_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49850_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49851_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49851_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49850_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49850_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49851_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49851_Cy5 560_Cy3 590 Cy5.gel

File 49850_Cy5 560_Cy3 590 Cy3.gel had 97818 saturated pixels of 5304000

File 49851_Cy5 560_Cy3 590 Cy3.gel had 69973 saturated pixels of 5304000

Starting scan of Strip1: 49852 Strip2: 49853

Finished scan of Strip1: 49852 Strip2: 49853

Scanned images in 00:31:18.1004212:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49852_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49852_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49853_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49853_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49852_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49852_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49853_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49853_Cy5 560_Cy3 590 Cy5.gel

File 49852_Cy5 560_Cy3 590 Cy3.gel had 81429 saturated pixels of 5304000

File 49853_Cy5 560_Cy3 590 Cy3.gel had 82430 saturated pixels of 5304000

Starting scan of Strip1: 49854 Strip2: 49855

Finished scan of Strip1: 49854 Strip2: 49855

Scanned images in 00:31:19.8435209:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49854_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49854_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49855_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49855_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49854_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49854_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49855_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49855_Cy5 560_Cy3 590 Cy5.gel

File 49854_Cy5 560_Cy3 590 Cy3.gel had 89075 saturated pixels of 5304000

File 49855_Cy5 560_Cy3 590 Cy3.gel had 71489 saturated pixels of 5304000